Platelet derived growth factor (PDGF) is a potent proliferative agent in cells of mesenchymal origin (Antoniades, H. N. et al. (1979) Proc. Natl. Acad. Sci. USA 76:1809–1813; Bowen-Pope, D. F. and Ross, R. (1982) J. Biol. Chem. 257:5161–5171; Heldin, C-H. et al. (1983) J. Biol. Chem. 258:10054–10059, all of which are incorporated herein by reference). PDGF (M.W. 30 KDa) is a disulfide-linked dimer consisting of 2 homologous chains termed A or B (Johnsson, A. et al. (1982) Biochem. Biophys. Res. Commun. 104:66–74, which is incorporated herein by reference). The chains may combine with chains of the same or the other type, resulting in 3 isoforms AA, BB or AB (Heldin, C-H. et al. (1986) Nature 319:511–514, which is incorporated herein by reference). The mitogen PDGF was first identified (Antoniades, H. N. (1979) Proc. Natl. Acad. Sci. USA 76:1809–1813; Raines, E. W. and Ross, R. (1982) J. Biol. Chem. 257:5154–5160, both of which are incorporated herein by reference) and purified from human platelets (Raines, op. cit.), though subsequent research has shown that several cell types including vascular endothelial cells, vascular smooth muscle cells, macrophages and even fibroblasts synthesize PDGF (Ross, R. et al. (1986) Cell 46:155–169, which is incorporated herein by reference).
The cellular proliferation induced by all isoforms of PDGF is mediated by ligand binding to the PDGF receptor (Heldin, C-H. (1983) op. cit., Ek, B. et al. (1982) Nature 295:419–420; Glenn, K. et al. (1982) J. Biol. Chem. 257:5172–5176; Frackelton, A. R. et al. (1984) J. Biol. Chem. 259:7909–7915; Williams, L. T. et al. (1984) J. Biol. Chem. 259:5287–5294, all of which are incorporated herein by reference). The PDGF receptor (M.W. 180 KDa) belongs to the tyrosine kinase family and consists of two receptor subtypes, termed type A (or type alpha) (Matsui, T. et al. (1989) Science 243:800–804, and Claesson-Welsh, L. (1989) Proc. Natl. Acad. Sci. USA 86:4917–4921, both of which are incorporated herein by reference) and type B (or type beta) (Yarden, Y. et al. (1986) Nature 323:226–232, and Escobedo, J. A. et al. (1988) Science 240:1532–1534, both of which are incorporated herein by reference).
High affinity binding of PDGF to the receptor is followed by receptor dimerization (Bishayee, S. et al. (1989) J. Biol. Chem. 264:11699–11705, and Heldin, C-H. et al. (1989) J. Biol. Chem. 264:8905–8912) and autophosphorylation (Frackelton, et al. op. cit.), and results in a complicated series of intracellular signalling events culminating in DNA synthesis. Mouse and human PDGF beta receptor and PDGF alpha receptor genes have been cloned (Matsui et al. op. cit., Claesson-Welsh et al. op. cit., Yarden et al. op. cit., and Escobedo et al. op. cit.). When referring to PDGF receptors herein, type A and type alpha or α-PDGFR are used interchangeably, as are type B and type beta or β-PDGFR.
The two receptor isoforms may be distinguished by their markedly different ligand binding specificities. PDGF beta receptor binds only B-chain (isoforms BB and AB), while PDGF alpha receptor can bind all forms of PDGF (isoforms containing A and/or B chain (Matsui et al. op. cit., Claesson-Welsh et al. op. cit., and Seifert, R. A. et al. (1989) J. Biol. Chem. 264:8771–8778). The PDGF receptor shows a high degree of structural homology to the macrophage-colony stimulating factor receptor (Coussens, L. et al. (1986) Nature 320:277–280) and the c-kit protooncogene product (Yarden, et al., op. cit.).
The PDGF receptors are characterized by an extracellular domain which may be demarcated into five Ig-like domains (Domains or D 1-5) based on their β-sheet rich structure. These Ig repeats of approximately 100 amino acids each have regularly spaced cysteine residues (except in the fourth repeat). The receptor has a single transmembrane domain and a cytoplasmic tyrosine kinase domain (Williams, L. T. (1989) Science 243:1564–1570, which is incorporated herein by reference).
PDGF plays an important role during normal physiological processes such as tissue repair and embryogenesis (Ross, R. et al. op. cit.). However, studies now implicate this potent mitogen in pathological proliferative disorders and in the development of certain carcinomas (Ross, R. et al. op. cit.). Expression of PDGF A chain and PDGF beta receptor has been detected in human atherosclerotic plaques by in situ hybridization (Wilcox, J. N. et al. (1988) J. Clin. Invest. 82:1134–1143). Recently, Ferns et al. ((1991) Science 253:1129–1132) have reported that a polyclonal antibody to PDGF significantly reduced the formation of intimal lesions in deendothelialized carotid arteries of athymic nude rats. PDGF has been implicated in the pathology of proliferative diseases in cells of mesenchymal origin (Nister, M. et al. (1984) Proc. Natl. Acad. Sci. USA 81:926–930, and Nister, M. et al. (1987) Cancer Res. 47:4953–4961, both of which are incorporated herein by reference). Golden et al. have reported that PDGF A chain message was increased in areas of intimal hyperplasia in a baboon model for vascular grafts ((1990) J. Vasc. Surg. 11:580–585). PDGF is also chemotactic for smooth muscle (Westermark, B. et al. (1990) Proc. Natl. Acad. Sci. USA 87:128–132), and platelet PDGF may be the causative agent for the migration and proliferation of smooth muscle cells in the ballooned rat carotid artery, which results in significant stenosis.
The study of other growth factors and their receptors has been aided by the invention of antibodies against the receptors. For example, antibodies that recognize the epidermal growth factor receptor have proved to be powerful tools in evaluating the mechanism of receptor activation (Spaargaren, M. et al. (1991) J. Biol. Chem. 266:1733–1739, which is incorporated herein by reference). Antibodies against receptors for interleukin-2 (IL-2) inhibit IL-2 internalization, and thus inhibit the subsequent induction of proliferation of responsive cells (Duprez, V. et al. (1991) J. Biol. Chem. 1497–1501, which is incorporated herein by reference). Similarly, a monoclonal antibody against the epidermal growth factor (EGF) receptor inhibits estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 (Eppstein, D. A. (1989) J. Cell. Physiol. 141:420–430, which is incorporated herein by reference). Such antibodies may be of great therapeutic value in treating growth factor-mediated diseases.
Several groups have isolated antibodies against PDGF receptors, but these antibodies have limited utility (see, for example, Kawahara, R. S. et al. (1987) Biochem. Biophys. Res. Commun. 147:839–845, which is incorporated herein by reference). Additional monoclonal antibodies have been raised against the extracellular PDGF-binding domain of a PDGF receptor from porcine uterus (Ronnestrand, L. and Terracio, L. (1988) J. Biol. Chem. 263:10429–10435, which is incorporated herein by reference), but these antibodies did not inhibit binding of 125I-labelled PDGF to human fibroblasts. Numerous antibodies against a PDGF receptor that did not inhibit PDGF activity have also been reported by Kanakaraj, P. S. et al. (1991) Biochemistry 30:1761–1767; Claesson-Welsh, L. et al. (1989) J. Biol. Chem. 264:1742–1747; Seifert, R. A. et al. (1989) J. Biol. Chem. 264:8771–8778; Kumjian, D. A. et al. (1989) Proc. Natl. Acad. Sci. USA 86:8232–8236; Bishayee, S. et al. (1988) Mol. Cell. Biol. 8:3696–3702; Hart, C. E. et al. (1987) J. Biol. Chem. 262:10780–10785; Escobedo, J. A. et al. (1988) J. Biol. Chem. 263:1482–1487; Daniel, T. O. et al. (1987) J. Biol. Chem. 262:9778–9784; Keating, M. T. and L. T. Williams (1987) J. Biol. Chem. 262:7932–7937; Kazlauskas, A. and J. A. Copper (1990) EMBO J. 9:3279–3286; all of which are incorporated herein by reference.
Thus, there exists a need for immunoglobulin and other agents capable of specifically inhibiting activation of the human receptor and/or proliferation of cells displaying the human type beta PDGF receptor. Such agents would be useful in mapping the different functional domains of the receptor, and in dissecting the role of PDGF and its receptors in normal and disease processes. Furthermore, such agents will have therapeutic value in the treatment of PDGF-mediated proliferative diseases, and also diseases involving PDGF-mediated chemotaxis and migration. Such diseases include:
a) restenosis, including coronary restenosis after angioplasty, atherectomy, or other invasive methods of plaque removal, and renal or peripheral artery restenosis after the same procedures;
b) vascular proliferative phenomena and fibrosis associated with other forms of acute injury such as: pulmonary fibrosis associated with adult respiratory distress syndrome, renal fibrosis associated with nephritis, coronary stenosis associated with Kawasake's disease, and vascular narrowings associated with other arteritides such as Takayasha's disease;
c) prevention of narrowings in vein grafts;
d) prevention of narrowings due to accelerated smooth muscle cell migration and proliferation in transplanted organs;
e) other fibrotic processes, such as scleroderma, myofibrosis; and
f) inhibition of tumor cell proliferation which is mediated by PDGF.
The present invention fulfills these and other needs.